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Oleic Acid Glucose-Independently Stimulates Glucagon Secretion by Increasing Cytoplasmic Ca²⁺ via Endoplasmic Reticulum Ca²⁺ Release and Ca²⁺ Influx in the Rat Islet -Cells

Division of Integrative Physiology (K.F., F.M., K.D., M.N., T.Yad.), Department of Physiology, and Division of Histology and Cell Biology (K.F., T.Yas.), Department of Anatomy, Jichi Medical University, School of Medicine, Shimotsuke, Tochigi 329-0498, Japan

Address all correspondence and requests for reprints to: Dr. T. Yada, Department of Physiology, Jichi Medical University, School of Medicine, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail: tyada{at}jichi.ac.jp.

	Abstract

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The effect of long-chain free fatty acids on glucagon secretion from islet -cells has been a controversial issue. This study examined direct effects of oleic acid (OA) on glucagon release from rat pancreatic islets and on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in single -cells by fura-2 fluorescence imaging. OA at 30 µM increased glucagon release from isolated islets in the presence of low (2.8 mM) and elevated (8.3 mM) glucose concentrations. OA at 6–10 µM concentration-dependently increased [Ca²⁺]_i in -cells, irrespective of glucose concentrations (1.4, 2.8, and 8.3 mM). OA at 10 µM increased [Ca²⁺]_i in 90% of -cells. OA-induced [Ca²⁺]_i increases were strongly inhibited by the endoplasmic reticulum Ca²⁺-pump inhibitors cyclopiazonic acid and thapsigargin and by 2-aminoethoxydiphenyl borate, the blocker of both inositol 1,4,5-trisphosphate receptors and store-operated Ca²⁺ channels. Furthermore, the amplitude, but not incidence, of OA-induced [Ca²⁺]_i increases was reduced substantially by Ca²⁺-free conditions and mildly by an L-type Ca²⁺ channel blocker, nitrendipine, and an ATP-sensitive K⁺ channel activator, diazoxide. OA-induced glucagon release was also inhibited mildly by nitrendipine and strongly by 2-aminoethoxydiphenyl borate. These results indicate that OA glucose-independently stimulates glucagon release by increasing [Ca²⁺]_i in rat pancreatic -cells and that the [Ca²⁺]_i increase is triggered by Ca²⁺ release from endoplasmic reticulum and amplified by Ca²⁺ influx possibly via store-operated channels and via voltage-dependent L-type Ca²⁺ channels. The glucose-independent action of OA to stimulate glucagon release from -cells may operate under hypoglycemic conditions when plasma free fatty acids levels are elevated, possibly playing a role in maintaining glucose metabolism.

	Introduction

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INTERACTION BETWEEN nutrients (glucose, fatty acids, and amino acids) and the endocrine system plays an essential role in energy homeostasis, in which insulin and glucagon are key hormones. Long-chain free fatty acids (FFAs), as well as glucose, are primary energy sources for the body. FFAs are also considered one of systemic signals that link metabolic states to the endocrine pancreas. In numerous studies, both stimulatory and inhibitory effects of FFAs on insulin secretion have been documented. Acute increases in circulating FFAs in the short term promote insulin secretion from pancreatic ß-cells (1, 2, 3, 4, 5). On the other hand, when the plasma level of FFAs is chronically elevated, it inhibits insulin secretion (6, 7, 8), a phenomenon recognized as the lipotoxicity.

Conversely, the effect of FFAs on glucagon secretion is less understood compared with insulin secretion. Earlier studies established the theory that FFAs inhibit glucagon secretion. Elevation of circulating FFAs suppresses glucagon secretion in animals (9) and humans (10, 11, 12). This could possibly be due either to direct FFA effects or to paracrine effects of hormones released by FFAs. On the other hand, it has recently been reported that palmitic acid stimulates glucagon secretion from mouse pancreatic -cells (13, 14). Thus, the effect of FFAs on glucagon secretion is a controversial issue. Moreover, signal transduction mechanisms of FFA in -cells are little known.

This study employed oleic acid (OA) because this FFA exists abundantly in the plasma of rats (15). The effect of OA on glucagon secretion from isolated rat islets was examined. It is generally accepted that glucagon release is initiated by an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in -cells; experimental conditions known to stimulate glucagon secretion are associated with increased [Ca²⁺]_i (16, 17, 18). Hence, we studied direct effects of OA on [Ca²⁺]_i in single -cells isolated from rats by fura-2 fluorescence imaging and explored underlying signaling mechanisms.

	Materials and Methods

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Solutions and chemicals
Measurements were carried out in HEPES and Krebs-Ringer bicarbonate buffer (KRBH) composed of 129 mM NaCl, 5.0 mM NaHCO₃, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.0 mM CaCl₂, 1.2 mM MgSO₄, and 10 mM HEPES (pH 7.4) supplemented with 0.01% BSA fraction V (Roche, Penzberg, Germany). Fetal bovine serum was from Equitec-Bio (Kerrville, TX). Collagenase type XI, 2-aminoethoxydiphenyl borate (2-APB), diazoxide, and thapsigargin were from Sigma Chemical Co. (St. Louis, MO). Fura-2/acetoxymethylester and fura-2 free acid were from Dojin Chemical (Kumamoto, Japan). Calcium calibration buffer kit no. 2 was from Molecular Probes (Leiden, The Netherlands). All other chemicals were from Wako Pure Chemicals (Osaka, Japan).

Isolation of islets and single islet cells and culture of single islet cells
Wistar rats aged 10–12 wk (SLC, Hamamatsu, Japan) were deeply anesthetized with an ip injection of 25% carbamic acid ethyl ester (5 ml/kg body weight). Isolation of islets and their dispersion into single islet cells were carried out as previously reported (19, 20). Briefly, 1 mg/ml collagenase XI dissolved in 5 mM Ca²⁺-containing KRBH was injected into the common bile duct at the distal end. The pancreas was removed and incubated at 37 C for 16 min. Islets were hand collected under a microscope. The isolated islets were either used for the secretion study or further dispersed into single cells by incubation with Ca²⁺-free KRBH containing 1 mM EGTA. The single cells were plated on coverslips and cultured for 1 d in MEM containing 5.6 mM glucose, 100 µg/ml streptomycin, 100 U/ml penicillin, and 10% fetal bovine serum at 37 C in 95% air with 5% CO₂.

Preparation of solutions containing OA and indication of OA concentrations
OA was stocked at 10 mM in dimethylsulfoxide, and an aliquot of the stock solution was added to KRBH to achieve the final concentrations indicated in the text, which indicate the concentrations of total, free plus bound, OA. To avoid significant binding of FFAs to BSA reported previously (see reviews in Refs. 21 and 22), all experiments in the present study were carried out in KRBH containing BSA at a concentration as low as 0.01%, unless otherwise indicated. It is difficult to experimentally determine the accurate concentration of FFA under fixed total FFA and BSA concentrations. Furthermore, although several methods to calculate the concentration of FFA are available (23, 24, 25), they can only estimate it. In this study, therefore, instead of calculating the estimated concentrations of free OA, we indicate the total OA concentrations together with a fixed BSA concentration (0.01%), by which experimental conditions can be stably reproduced.

Measurements of glucagon and insulin release
Groups of 10 islets were incubated at 37 C for 0.5 h in KRBH with 2.8 mM glucose and 0.01% BSA and then for 0.5 h in KRBH with 2.8 or 8.3 mM glucose and 0.01% BSA. Glucagon and insulin concentrations were determined using an ELISA kit (Wako) and enzyme immunosorbent assay kits (Morinaga, Yokohama, Japan), respectively.

Measurements of [Ca²⁺]_i in -cells
Cytoplasmic [Ca²⁺]_i was measured by dual-wavelength fura-2 micofluorometry and digital imaging as previously reports (19, 20). Single cells on coverslips were incubated with 1 µM fura-2/acetoxymethylester for 30 min at 37 C in KRBH containing 2.8 mM glucose. Cells were then mounted in a chamber and superfused with KRBH at a rate of 1 ml/min at 37 C. The fura-2-loaded cells were excited at 340 and 380 nm alternately, the emission signals at 510 nm were detected every 5 or 10 sec by a cooled charge-coupled device camera, and the ratio was produced by an AquaCosmos system (Hamamatsu Photonics, Hamamatsu, Japan). Ratio values were converted to [Ca²⁺]_i according to calibration curves obtained from the relationship between free Ca²⁺ concentration and the ratio determined in a cytosol-mimicking solution using a calcium calibration buffer kit and fura-2 free acid. When increases in [Ca²⁺]_i took place within 5 min after addition of agents and their amplitudes were 30 nM or larger, they were considered responses.

Immunocytochemistry of single islet cells and correlation of [Ca²⁺]_i and immunocytochemical data
After measurements of [Ca²⁺]_i, -cells were identified by subsequent immunocytochemical staining using an antiglucagon antiserum. The cells on coverslips were fixed with 4% paraformaldehyde in PBS for 30 min. They were washed with PBS and then treated with 1% normal horse serum and 0.4% Triton X-100 in PBS for 1 h. Antiglucagon rabbit polyclonal antibodies (final dilution, 1:1000; DakoCytomation, Glostrup, Denmark) were used as primary antibody. Cells were incubated with antiglucagon antiserum at room temperature for 16 h and then with biotinylated antirabbit IgG (Vector Laboratories, Inc., Temecula, CA). Immunoreactivity was visualized with diaminobenzidine after labeling with streptavidin-conjugated horseradish peroxidase (DakoCytomation).

Correlation of the [Ca²⁺]_i and immunocytochemical data was carried out as previously reported (19, 20); the phase-contrast photographs of islet cells on coverslips in the microscopic field taken at the end of [Ca²⁺]_i measurements were compared with the photographs of islet cells on coverslips after immunocytochemical staining. [Ca²⁺]_i data were obtained only from the immunocytochemically identified -cells.

Statistical analysis
The data are presented as the mean ± SE (n = number of experiments or cells). Each study was based on -cells prepared from at least three rats. Statistical analysis was performed using Fisher’s protected least significant difference. P values < 0.05 were considered statistically significant.

	Results

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OA stimulates glucagon secretion from isolated pancreatic islets
Isolated islets were challenged with various concentrations of OA under static incubation conditions, followed by measurements of glucagon release by enzyme-linked immunosorbent assay. As shown in Fig. 1A, in the presence of 2.8 mM glucose, OA increased glucagon secretion from islets in a concentration-dependent manner; at 30 and 50 µM, it significantly (P < 0.05) increased glucagon release (in pg/ml·islet·0.5 h: 181 ± 12.6 with 30 µM OA and 189 ± 11.1 with 50 µM OA vs. 92.7 ± 7.3 in control).

View larger version (16K): [in this window] [in a new window]   FIG. 1. Effects of OA on glucagon secretion from rat islets. A, OA at 30 and 50 µM, but not 10 µM, significantly increased glucagon secretion from isolated islets in the presence of 2.8 mM glucose. **, P < 0.01 vs. 2.8 mM glucose alone. B, Glucagon secretion was lower and insulin secretion was higher in the presence of 8.3 mM glucose (8.3G) than in 2.8G. In 8.3G, OA at 30 µM significantly increased glucagon secretion (8.3G+OA). *, P < 0.05; **, P < 0.01 vs. 8.3 mM glucose alone. The results are presented as the mean ± SE for eight to 26 experiments in each group.

OA increases [Ca²⁺]_i in single pancreatic -cells
Single islet cells were superfused with KRBH containing 2.8 mM glucose and subjected to [Ca²⁺]_i measurements with fura-2 fluorescence imaging. OA at 10 µM induced large increases in [Ca²⁺]_i in a single islet cell, which was subsequently proved to be immunoreactive to glucagon (Fig. 2A). The result demonstrated that OA directly interacts with rat -cells to increase [Ca²⁺]_i. In some OA-responsive -cells, [Ca²⁺]_i increased in an oscillatory pattern (Fig. 2B). Repeated administration of OA induced repetitive [Ca²⁺]_i increases (Fig. 2, A and B). OA (10 µM) increased [Ca²⁺]_i with latency of 99 ± 3.5 sec (209 cells) after addition to the superfusion medium and with amplitude of 151.4 ± 5.3 nM (209 cells).

View larger version (14K): [in this window] [in a new window]   FIG. 2. OA induced [Ca2+]i increase in single pancreatic -cells. A, In 2.8 mM glucose, OA at 10 µM evoked increases in [Ca2+]i in a single islet cell, which was proved to be immunoreactive to glucagon by subsequent immunocytochemical staining with antiglucagon antiserum. Scale bar, 10 µm. The bars above the tracing specify the periods of exposure to compounds indicated. B, OA occasionally induced [Ca2+]i oscillations in -cells. C, Elevation of BSA concentration to 0.1% inhibited the [Ca2+]i response to 10 µM OA. BSA concentration in superfusion solutions was 0.01% in all other experiments.

Concentration-dependent and glucose-independent ability of OA to increase [Ca²⁺]_i
OA at 4 µM increased [Ca²⁺]_i in none of 43 -cells, at 6 µM in four of 53 (8%), at 8 µM in 51 of 95 (54%), and at 10 µM in 209 of 233 (90%), showing a concentration-dependent effect (Fig. 3, A and B). OA at 8 µM evoked oscillatory [Ca²⁺]_i increases in eight of 51 OA-responding -cells (16%) and at 10 µM in 56 of 209 cells (27%). The amplitude of OA-evoked [Ca²⁺]_i increase above the basal level ([Ca²⁺]_i) was integrated over the 5-min administration period and expressed by the averaged amount per minute. OA increased the integrated [Ca²⁺]_i in a concentration-dependent manner (Fig. 3C). OA at 10 µM induced large increases in [Ca²⁺]_i in as much as 90% of -cells. Therefore, for the rest of study, [Ca²⁺]_i measurements were performed using 10 µM OA. When the concentration-response relationships were compared, the concentration of OA required for glucagon release was three to five times higher than that for [Ca²⁺]_i increase in -cells. We previously also reported that the OA concentration needed for insulin release was five to 10 times higher than that for [Ca²⁺]_i increase in ß-cells (26). This apparent discrepancy in the effective concentration for two parameters may be due to the different experimental conditions employed: [Ca²⁺]_i was measured in single cells, whereas glucagon/insulin release was measured in islets. In islet experiments, because OA is a hydrophobic molecule, its actual concentration within islets may decline, and hence a higher concentration of OA might have to be added.

View larger version (14K): [in this window] [in a new window]   FIG. 3. Concentration-dependent and glucose-independent effects of OA to increase [Ca2+]i. A, OA at 8 and 10 µM increased [Ca2+]i, whereas OA at 6 µM had little effect on [Ca2+]i in the presence of 2.8 mM glucose. B, Percentage of -cells responding to 4, 6, 8, and 10 µM OA in the presence of 2.8 mM glucose. The numbers above each point indicate the number of cells that responded over the number of cells examined. C, The increases in [Ca2+]i during stimulation with 6, 8, and 10 µM OA for 5 min was integrated and averaged for the cells examined. *, P < 0.01 vs. 6 µM OA. D and E, OA increased [Ca2+]i independently of glucose concentrations. Neither lower glucose concentration (1.4 mM) (D) nor higher glucose concentration (8.3 mM) (E) affected 10 µM OA-induced [Ca2+]i increases.

Endoplasmic reticulum (ER) Ca²⁺ pump blockers inhibited [Ca²⁺]_i responses to OA
To determine the mechanisms underlying OA-induced Ca²⁺ increases, we used inhibitors of various Ca²⁺ signaling pathways. We confirmed the involvement of intracellular Ca²⁺ stores in OA signaling by blocking sarcoplasmic/endoplasmic Ca²⁺-ATPase (SERCA) pumps with two structurally different inhibitors: cyclopiazonic acid (CPA) and thapsigargin (Tg) (20, 28). OA-induced [Ca²⁺]_i increase in -cells was virtually abolished in the presence of 50 µM CPA (Fig. 4A), whereas 10 mM L-arginine- and 25 mM KCl-induced [Ca²⁺]_i increases were unaffected (Fig. 4B). Tg at 1 µM also inhibited OA-induced [Ca²⁺]_i increases in -cells (Fig. 4C). Furthermore, OA-induced [Ca²⁺]_i increases were inhibited by 100 µM 2-APB, an inositol 1,4,5-trisphosphate (IP3) receptor antagonist that also reportedly blocks store-operated Ca²⁺ influx (Fig. 4D). In the presence of CPA, Tg, or 2-APB, only 11 of 52 -cells (21%), five of 42 (12%), or 15 of 65 (23%), respectively, exhibited [Ca²⁺]_i responses to 10 µM OA (Fig. 4F). Moreover, the integrated [Ca²⁺]_i during exposure to OA in the presence of CPA, Tg, or 2-APB was markedly reduced compared with control (in nM/min: 40.3 ± 12.8 (52 cells) with CPA, 7.4 ± 6.1 (42 cells) with Tg, and 48.2 ± 4.8 (65 cells) with 2-APB vs. 307.7 ± 13.9 (233 cells) in control; P < 0.01; Fig. 4G). In contrast, the presence of ryanodine (10 µM) did not inhibit OA-induced [Ca²⁺]_i increases [471.4 ± 46.1 nM/min, 27 of 29 cells (93%), Fig. 4, E–G].

View larger version (15K): [in this window] [in a new window]   FIG. 4. Effects of ER Ca2+ pump blockers on [Ca2+]i responses to OA. A, Treatment with 50 µM cyclopiazonic acid (CPA), SERCA blocker, inhibited the 10 µM OA-induced [Ca2+]i increases. B, On the other hand, [Ca2+]i increases induced by 10 mM L-arginine (Arg) and 25 mM KCl were not affected by CPA. C, Another blocker of SERCA, 1 µM Tg, also inhibited OA-induced [Ca2+]i increase. D, OA-induced [Ca2+]i increase was markedly inhibited by 100 µM 2-APB, an antagonist of IP3 receptor and store-operated Ca2+ influx. E, The presence of 10 µM ryanodine had little effect on OA-induced [Ca2+]i increase. F, Percentage of -cells responding to 10 µM OA in the presence of CPA, Tg, 2-APB, or ryanodine (Rya). The numbers above each bar indicate the number of -cells responding over that examined. G, The amplitude of [Ca2+]i increases during stimulation with OA, integrated and averaged for -cells examined, was significantly inhibited in the presence of CPA, Tg, and 2-APB but not Rya. *, P < 0.01 vs. control conditions.

View larger version (20K): [in this window] [in a new window]   FIG. 5. Effects of OA on Ca2+ influx from extracellular Ca2+. In Ca2+-free conditions, 10 µM OA-induced [Ca2+]i increase was attenuated but not completely abolished (A, D, and E). OA-induced [Ca2+]i increase was partially inhibited by 5 µM NTD, a blocker of L-type Ca2+ channels (B, D, and E). OA-induced [Ca2+]i increase was also partially inhibited by 100 µM diazoxide, an activator of KATP channels (C–E). D, Percentage of -cells responding to 10 µM OA under control and Ca2+-free conditions and in the presence of NTD, diazoxide, or -conotoxin GVIA. The numbers above each bar indicate the number of -cells responding over that examined. E, The amplitude of [Ca2+]i increases during stimulation with OA were integrated and averaged for -cells examined in these conditions. *, P < 0.01 vs. control conditions.

It is suggested that ATP-sensitive K⁺ (KATP) channels regulate the membrane potential in -cells as well as in ß-cells (29, 30, 31). Thus, activation of KATP channels is expected to hyperpolarize -cells and thereby inhibit the voltage-gated Ca²⁺ channels including L-type Ca²⁺ channels. In the present of a KATP channel activator, diazoxide (100 µM), the 10 µM OA-induced [Ca²⁺]_i increase was observed in 74 of 87 -cells (85%) (Fig. 5, C and D), but the integrated [Ca²⁺]_i during exposure to OA was reduced to a level similar to that obtained with NTD [223.0 ± 17.8 nM/min (87 cells), Fig. 5E].

OA-induced [Ca²⁺]_i increase was markedly inhibited in Ca²⁺-free conditions, whereas it was only mildly inhibited by blockade of voltage-gated Ca²⁺ channels. These results suggested the involvement of other Ca²⁺ influx pathways. A store-operated Ca²⁺ channel blocker, 2-APB (100 µM), which has also been known as an IP3 receptor antagonist, strongly inhibited OA-induced [Ca²⁺]_i increases in -cells [15 of 64 -cells (23%); 48.2 ± 4.8 nM/min; Fig. 4, D, F, and G].

Effects of inhibitors of OA-induced [Ca²⁺]_i increases on glucagon release
The increase in glucagon release in response to OA (30 µM) at 2.8 mM glucose was significantly attenuated by NTD (5 µM) (in pg/ml·islet·0.5 h: 110.2 ± 8.6 with NTD plus OA vs. 191.1 ± 17.7 with OA alone; P < 0.05; Fig. 6). Furthermore, 2-APB (100 µM) markedly reduced the OA-induced increase in glucagon release (59.3 ± 7.3 pg/ml·islet·0.5 h; P < 0.05; Fig. 6).

View larger version (14K): [in this window] [in a new window]   FIG. 6. Effects of inhibitors of OA-induced [Ca2+]i increases on glucagon release from rat islets. The increase in glucagon release in response to OA (30 µM) at 2.8 mM glucose was significantly attenuated by NTD (5 µM). Furthermore, 2-APB (100 µM) markedly reduced the OA-induced increase in glucagon release. *, P < 0.05; **, P < 0.01.

	Discussion

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It has been demonstrated that initiation of glucagon release is preceded by an increase in [Ca²⁺]_i (16, 17, 18). The present study showed that OA increased [Ca²⁺]_i in 90% of -cells. Moreover, both the OA-induced [Ca²⁺]_i increase and glucagon secretion took place in a glucose-independent manner and were inhibited by 2-APB and nitrendipine. These results suggest that the OA-induced [Ca²⁺]_i increase may lead to glucagon release in -cells. However, the concentrations of OA required for glucagon release were three to five times higher than those for [Ca²⁺]_i increases. Such a shift in effective concentrations was also observed for OA-induced insulin release and [Ca²⁺]_i increases in ß-cells in our previous study (26). This may be due to different experimental conditions employed; [Ca²⁺]_i was measured in single -cells, whereas glucagon release was measured in islets. In the former case, -cells are expected to see OA at the concentrations added in superfusion solutions. By contrast, islets are surrounded by a partial capsule of fibroblasts and collagen fibers (32), and -cells are located not only at the surface but also inside of the islet. Hence, the concentration of OA, a lipophilic molecule, is expected to decline within islets. Therefore, the actual concentrations of OA around -cells within islets may be lower than those originally added in solutions. If that is the case, higher concentrations of OA are required in the islet experiments than in the single-cell experiments to get comparable effects. An alternative possibility that unknown factors that are released from islets modulate the effects of OA on -cells in the islet experiments cannot be excluded.

In this study, the concentration of BSA in the superfusion solution was as low as 0.01%. When the BSA concentration was elevated to 0.1%, the [Ca²⁺]_i response to 10 µM OA was abolished. Previous studies by us and others also reported that elevation of the BSA concentration eliminated the ability of FFAs to increase [Ca²⁺]_i in pancreatic ß-cells (26) and to stimulate secretion of insulin (33) and glucagon-like peptide-1 (34). FFAs tightly bind to BSA (21, 22). Therefore, it is likely that the free, but not bound, form of OA interacts with pancreatic -cells to elicit [Ca²⁺]_i increases and glucagon release in our study.

OA-induced [Ca²⁺]_i increases were virtually abolished by SERCA pump blockers CPA and Tg and by 2-APB, an IP3 receptor antagonist that also reportedly blocks store-operated Ca²⁺ influx (35, 36). This suggests that OA-induced [Ca²⁺]_i increases depend on mobilization of Ca²⁺ from ER, which is mediated by IP3 receptor and/or associated with store-operated Ca²⁺ influx. In contrast, Ca²⁺-free conditions only mildly reduced the number of -cells that responded to OA, although it substantially decreased OA-induced [Ca²⁺]_i in -cells. The results suggest that OA increases Ca²⁺ influx, but it is not related to initiation of [Ca²⁺]_i increase. This Ca²⁺ influx was partially inhibited by the L-type Ca²⁺ channel blocker NTD. Furthermore, diazoxide, which is expected to open KATP channels and hyperpolarize the membrane (31), suppressed the amplitude of OA-evoked [Ca²⁺]_i increases to a level similar to that obtained with NTD. The N-type Ca²⁺ channel blocker, -conotoxin GVIA, had little effect on the OA-induced [Ca²⁺]_i increases. These results suggest that OA stimulates Ca²⁺ influx through voltage-dependent L-type Ca²⁺ channels. It should also be noted that Ca²⁺-free conditions reduced the amplitude of OA-evoked [Ca²⁺]_i increases to a much greater extent than NTD and diazoxide, indicative of a possible involvement of voltage-independent Ca²⁺ influx in OA-induced [Ca²⁺]_i increases. OA-induced [Ca²⁺]_i increases were strongly suppressed by 2-APB, a drug used as a blocker of store-operated Ca²⁺ influx (36). Liu et al. (37) reported that adrenaline stimulation and glucose inhibition of [Ca²⁺]_i increases in the mouse -cells involve modulation of a store-operated Ca²⁺ channel. Therefore, a substantial portion of the OA-induced [Ca²⁺]_i increase could result from Ca²⁺ influx through the store-operated Ca²⁺ channel, Taken together, our data indicate that the [Ca²⁺]_i increase is triggered by Ca²⁺ release from ER and amplified by voltage-independent Ca²⁺ influx possibly via store-operated channels and by voltage-dependent Ca²⁺ influx via L-type Ca²⁺ channels. Moreover, NTD partially and 2-APB strongly inhibited OA-induced glucagon secretion as well as [Ca²⁺]_i increase. These results suggest that OA-induced stimulation of glucagon secretion is mediated, at least partly, by the [Ca²⁺]_i increase in -cells. Recently, Olofsson and colleagues (13) reported that the saturated FFA palmitate stimulated glucagon secretion from intact mouse islets. They demonstrated that palmitate increases mouse -cell exocytosis principally by enhanced Ca²⁺ entry via L-type Ca²⁺ channels. Hence, some difference might exist between rat and mouse -cells in their Ca²⁺ signaling in response to FFAs. They suggested that palmitate-induced glucagon secretion requires metabolic conversion. Our previous study indicated that OA stimulates insulin release via G protein-coupled FFA receptor GPR40. OA evoked [Ca²⁺]_i increases in ß-cells with a latency of 44 ± 3 sec after its addition to superfusion medium (26). On the other hand, this study showed that OA evoked [Ca²⁺]_i increases in -cells with significant longer latency of 99 ± 4 sec. These findings suggest that OA increases [Ca²⁺]_i in -cells via a pathway not mediated by membrane receptors.

The amino acid L-arginine initiates glucagon secretion. Previous studies have indicated that L-arginine-induced glucagon release is a Ca²⁺-dependent mechanism (38), which results from membrane depolarization due to the electrogenic transport of this cationic amino acid (39) and consequent rise of [Ca²⁺]_i in -cells (18). In this study, unlike the case in OA, SERCA pump blockers had no effect on 10 mM L-arginine-induced [Ca²⁺]_i increases, whereas Ca²⁺-free conditions, as well as L-type Ca²⁺ channel blocker and KATP channel activator (Fujiwara, K., unpublished data), abolished them. These data confirmed that L-arginine-induced [Ca²⁺]_i increase is mediated by Ca²⁺ entry via voltage-dependent L-type Ca²⁺ channels (39). We also found that addition of L-arginine to the superfusion solution rapidly increased [Ca²⁺]_i with a latency of 16 ± 0.3 sec (n = 51), suggesting an instantaneous depolarization, whereas OA increased [Ca²⁺]_i with a much larger latency of 99 ± 3.5 sec, reinforcing that OA and L-arginine employ different Ca²⁺ signaling pathways.

When the plasma glucose concentration falls to levels around and less than 50 mg/dl (2.8 mM), an increase in glucagon secretion occurs (27). It is generally accepted that low glucose levels stimulate and high glucose levels inhibit glucagon secretion in in vivo situations. In the present study, however, neither lowering glucose concentration to 1.4 mM nor its elevation to 8.3 mM failed to alter [Ca²⁺]_i in single -cells. The result may indicate that glucose in this concentration range per se is not a direct regulator of -cells. On the other hand, glucagon secretion from isolated islets was moderately decreased by 8.3 mM glucose; this effect may be due to the inhibitory action of insulin, GABA, and/or zinc that are known to be released from glucose-stimulated ß-cells and could act on -cells via microcirculation or a paracrine route (40, 41, 42). In contrast, the present study demonstrated that the change in glucagon release from isolated islets by OA was much greater than that by glucose and that OA directly interacts with 90% of -cells to elicit large increases in [Ca²⁺]_i in the presence of both high and low glucose concentrations. These findings indicate that OA is a potent glucose-independent activator of rat pancreatic -cells.

Our observation indicated that OA directly activates -cells to release glucagon at low and high glucose concentrations, in contrast to the strictly glucose-dependent insulinotropic action of OA (26). The concentrations at which OA evoked these responses were in the physiological to pathophysiological range. These findings suggest that OA may serve as a glucagon secretagogue under physiological and/or pathophysiological conditions. Under conditions of hunger or fasting, the circulating level of FFAs increases, whereas glucose level is low; the elevated levels of FFAs glucose-independently stimulate secretion of glucagon, but not insulin, thereby mobilizing glucose to maintain the blood glucose level. FFAs could also play a pathological role in type 2 diabetes that shows both hyperlipidemia and hyperglycemia; the elevated levels of FFAs associated with hyperlipidemia might stimulate glucagon secretion, which further enhances hyperglycemia. However, additional studies are definitely needed to clarify the roles of FFA-induced glucagon release in the glucose metabolism and to further elucidate the molecular mechanisms of FFA-induced glucagon secretion in islet -cells.

	Acknowledgments

 
We are grateful to Dr. M. Tominaga (National Institutes of Natural Sciences, Okazaki) for technical advice and valuable discussion and thank Ms. Y. Nishizawa and S. Ohkuma for technical assistance.

	Footnotes

 
This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (K.F., T.Yad.), that on Priority Areas (15081101) (T.Yad.), a grant from the 21st century Center of Excellence program (T.Yad.), a grant from the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan (T.Yad.), an Insulin Research Award from Novo Nordisk Pharma Ltd. (T.Yad.), and a grant from Japan Diabetes Foundation (T.Yad.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 8, 2007

Abbreviations: 2-APB, 2-Aminoethoxydiphenyl borate; CPA, cyclopiazonic acid; ER, endoplasmic reticulum; FFA, free fatty acid; KATP, ATP-sensitive K⁺; KRBH, HEPES and Krebs-Ringer bicarbonate buffer; NTD, nitrendipine; OA, oleic acid; SERCA, sarcoplasmic/endoplasmic Ca²⁺-ATPase; TG, thapsigargin.

Received August 22, 2006.

Accepted for publication January 26, 2007.

	References

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